A Pan Plasmodium lateral flow recombinase polymerase amplification assay for monitoring malaria parasites in vectors and human populations

Robust diagnostic tools and surveillance are crucial for malaria control and elimination efforts. Malaria caused by neglected Plasmodium parasites is often underestimated due to the lack of rapid diagnostic tools that can accurately detect these species. While nucleic-acid amplification technologies stand out as the most sensitive methods for detecting and confirming Plasmodium species, their implementation in resource-constrained settings poses significant challenges. Here, we present a Pan Plasmodium recombinase polymerase amplification lateral flow (RPA–LF) assay, capable of detecting all six human infecting Plasmodium species in low resource settings. The Pan Plasmodium RPA-LF assay successfully detected low density clinical infections with a preliminary limit of detection between 10–100 fg/µl for P. falciparum. When combined with crude nucleic acid extraction, the assay can serve as a point-of-need tool for molecular xenomonitoring. This utility was demonstrated by screening laboratory-reared Anopheles stephensi mosquitoes fed with Plasmodium-infected blood, as well as field samples of An. funestus s.l. and An. gambiae s.l. collected from central Africa. Overall, our proof-of-concept Pan Plasmodium diagnostic tool has the potential to be applied for clinical and xenomonitoring field surveillance, and after further evaluation, could become an essential tool to assist malaria control and elimination.

www.nature.com/scientificreports/interventions has significantly reduced the malaria burden over the last two decades, an estimated 249 million cases and over half a million deaths still occur annually, with young children being the most affected 2 .In recent years many challenges, including, the emergence of drug and insecticide resistance, insufficient funding, and the effects of SARS-CoV-2 pandemic, have hindered malaria control progress, leading to a reversal in global elimination outcomes [3][4][5][6] .To overcome such challenges the development of new technologies is vital to aid and enhance existing control efforts.
A critical component of any malaria control strategy is pathogen monitoring and surveillance.This information is essential for guiding intervention efforts, identifying high-risk geographical regions or populations, providing outbreak warnings, and monitoring malaria transmission patterns 7 .Malaria transmission has commonly been determined by tracking the prevalence of Plasmodium parasites in the human population at a given time point (parasite rate; PR), using immunochromatographic-based rapid diagnostic tests (RDTs) or microscopy, which remains the gold-standard diagnostic method for malaria [8][9][10] .Xenomonitoring is a complementary approach, establishing the proportion of vectors harbouring a pathogen of interest, within a geographical region.Several techniques can be used for malaria xenomonitoring.One approach involves mosquito dissection by trained entomologists to confirm the presence of Plasmodium sporozoites, thereby assessing the mosquito's ability to transmit the disease.Additionally, molecular techniques such as polymerase chain reaction (PCR) can detect the presence of Plasmodium DNA in mosquitoes, regardless of the parasite stage.However, PCR alone does not indicate the mosquito's capacity to transmit malaria 11,12 .When combined with other metrics, such as the entomological inoculation rate (EIR)-the average number of infectious bites received per person-xenomonitoring can help assess the impact of intervention deployment on malaria infection dynamics and provide a comprehensive picture of ongoing disease transmission.
The accuracy and precision of both PR and EIR estimates depend on the efficacy of the methods used, particularly the ability to detect low-density infections in both human and Anopheles spp populations, which still contribute to malaria transmission.Nucleic-acid amplification technologies (NAATs), such as quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP), are the most sensitive techniques for detecting Plasmodium spp, outperforming other diagnostic methods like RDTs and microscopy [13][14][15] .However, NAATs typically require laboratory access, expensive equipment, and trained personnel, which are challenging to obtain in resource-limited settings.
To overcome these challenges, we sought to develop a Pan Plasmodium recombinase polymerase amplification (RPA)-based lateral flow (LF) assay ("Pan Plasmodium RPA-LF" assay).This assay aims to match the sensitivity of other NAATs while being suitable for use in low-resource field settings.It is designed to detect any of the six human malaria species (P.falciparum, P. vivax, P. malariae, P. knowlesi, P. ovale curtisi, and P. ovale wallikeri).The assay operates at a single temperature between 37-42 °C, which allows for the possibility of being powered by human body heat.The lower incubation temperature of our assay, compared to other isothermal NAATs such as LAMP (65 °C), is achieved using recombinase proteins (UvsX, UvsY).These proteins facilitate strand exchange between ssDNA primer oligos and dsDNA targets, eliminating the need for the denaturation and annealing steps typical in a standard PCR cycle 16 .Additionally, the assay reagents can be lyophilized, which improves shelf-life during storage and transportation, and removes the dependence on a cold chain, which can be unreliable in resource-limited settings.
In this work, we evaluate the Pan Plasmodium RPA-LF assay using a combination of clinical samples and both laboratory-reared and field-collected Anopheles spp mosquitoes.This provides a proof-of-concept assessment for the assay's use as a multifaceted surveillance tool.Moreover, this study demonstrates the viability of a crude nucleic acid extraction approach in tandem with the Pan Plasmodium RPA-LF assay, enhancing its applicability for point-of-need molecular xenomonitoring in resource-limited settings.

Assay design & in silico screening
The Pan Plasmodium RPA-LF assay was specifically designed for this study following established guidelines to detect all six human-pathogenic species of malaria: P. falciparum, P. vivax, P. knowlesi, P. malariae, P. ovale curtisi, and P. ovale wallikeri 17 .It targets the mitochondrial rRNA encoding region of Plasmodium spp, which encompasses a total of 5 genes (Pf3D7 Orthologs: PF3D7_MIT03800, PF3D7_MIT03900, PF3D7_MIT04000, PF3D7_MIT04100, PF3D7_MIT04200; sourced from PlasmoDB).Inclusivity of the assay was assessed in silico against a dataset of 890 publicly available mitochondrial sequences from all human-infecting Plasmodium species (see Supplementary Information).All oligonucleotide binding sites were conserved across 99.4% (885/890) of sequences screened (Supplementary Table 1).In addition, the specificity of the Pan Plasmodium RPA-LF assay was assessed in silico against the nt database using the NCBI Blastn tool.No alternative binding sites were identified in the human host (Homo sapiens (TaxID: 9606)) or vector (Anopheles spp (TaxID: 7164)), with 91.8% of identified hits originating from sequences classified under Plasmodiidae (see Supplementary Table 2).

Assay inclusivity and limit of detection (LoD)
Following in silico assessment, the Pan Plasmodium RPA-LF assay was first validated across all six human infecting Plasmodium species.For P. falciparum (Dd2) and P. knowlesi (A1-H.1),DNA extracted from cultured strains was utilised.For the other unculturable Plasmodium species (e.g., P. vivax, P. malariae, P. ovale curtisi and P. ovale wallikeri), DNA was extracted from clinical isolates, kindly provided by the UK Health Security Agency-Malaria Reference Laboratory (UKHSA-MRL, LSHTM), was used.All species were successfully detected (Fig. 1A, Supplementary Fig. 1), and the results were compared with qPCR performed in tandem (see Methods section).Cultured Plasmodium isolates had the lowest qPCR Ct values, with P. falciparum at 11.75 and P. knowlesi at 11.63, compared to clinical isolates which had a Ct range of 17.59-18.78,reflecting the high parasitaemia achievable in culture.Following inclusivity screening, the sensitivity of the Pan Plasmodium RPA-LF assay was assessed against P. falciparum.The use of parasite cultures ensured that only parasite DNA was present, unlike clinical isolates which contain a mix of parasite and human DNA from white blood cells.This facilitated accurate fluorescent-based quantification of P. falciparum using the Qubit high sensitivity DNA detection assay.Initial screening established the limit of detection (LoD) for the Pan Plasmodium RPA-LF assay against P. falciparum to be between 100 fg DNA/µl (10/10) and 10 fg DNA/µl (9/10) (Fig. 1B, Supplementary Fig. 2).
Tandem qPCR screening of the same isolates indicated that the failed detection of a single P. vivax isolate was likely due to its concentration being below the LoD of the Pan Plasmodium RPA-LF assay, with a Ct > 31, indicative of a Plasmodium spp.concentration less than 1 fg/µl as established in Fig. 1 (Supplementary Table 4).Additionally, one RPA-LF positive P. vivax isolate tested negative via tandem qPCR screening.

Xenomonitoring Proof-of-Concept
To evaluate the assay's potential for xenomonitoring applications, eight infected blood-fed and two uninfected Anopheles stephensi mosquitoes were provided by the Human Malaria Transmission Facility at LSHTM.Following DNA extraction using the Qiagen DNeasy Blood and Tissue Kit, all 10 vectors were screened with the Pan Plasmodium RPA-LF assay (Fig. 3).Six out of the eight infected blood-fed An. stephensi mosquitoes tested positive with the Pan Plasmodium RPA-LF assay.The two remaining infected mosquitoes (AS2, AS3) had parasite densities below the assay's limit of detection, as indicated by qPCR Ct values of 33.92 and 32.62, respectively, as established in Fig. 1.Both uninfected An. stephensi mosquitoes were negative in both RPA and qPCR assays.Subsequently, the Pan Plasmodium RPA-LF assay was assessed on 29 Anopheles spp.field samples, a mixture of An. funestus s.l. and An.gambiae s.l., collected between 2018 and 2019 from the Democratic Republic of the Congo (DRC).Tandem qPCR screening identified four out of the 29 field samples as positive (Ct value range: 25.45-30.31),consistent with previous molecular assessments conducted upon sample collection (Supplementary Table 6).However, the Pan Plasmodium RPA-LF assay successfully detected only two of these samples (Ct: 25.45, 27.23); the other two samples had Ct values (27.73, 30.31) below the assay's limit of detection.Among the two RPA-LF positive samples, only one had evidence of a recent blood meal, indicated by the presence of blood in the abdomen.The remaining 24 samples were negative by both qPCR and the Pan Plasmodium RPA-LF assay, aligning with prior molecular assessments (Supplementary Table 6).

Crude nucleic acid extraction
Most malaria surveillance methods in molecular xenomonitoring typically involve transporting collected samples to a central facility for sorting, morphological identification, pooling, and processing-often via dissection or nucleic acid extraction-before screening 20 .To enable the use of the Pan Plasmodium RPA-LF assay directly at  the point of collection, we have developed a room temperature (22 °C), polyethylene glycol (PEG)-enhanced chemical crude extraction method.This approach eliminates the need for specialised equipment and involves an alkaline lysis buffer based on potassium hydroxide, followed by neutralisation with a tris-acetate buffer (details in the Methods section) 21 .Following crude extraction, the sample can be directly introduced into the Pan Plasmodium RPA-LF assay.We initially validated this extraction method using eight individual An.stephensi mosquitoes, each recently fed on infected blood and provided by the Human Malaria Transmission Facility at LSHTM.To ensure complete submersion, we employed 75 µl of lysis buffer and 25 µl of neutralization buffer per mosquito.Following extraction, all eight mosquitoes tested positive for infection upon screening (Fig. 4, Supplementary Table 7).Subsequently, we created ten pools of mosquitoes: eight pools contained one infected-blood-fed An. stephensi each, along with four unfed An. stephensi, while two negative control pools consisted of five unfed mosquitoes each.Pools containing infected-blood-fed vectors consistently tested positive in the Pan Plasmodium RPA-LF assay following crude nucleic acid extraction using 150 µl of lysis buffer and 50 µl of neutralisation buffer (Fig. 4, Supplementary Table 7).

Discussion
In this study, we present a comprehensive Pan Plasmodium RPA-LF-based assay for disease surveillance, capable of detecting all six human-infecting Plasmodium species from clinical and cultured isolates.Our findings expand on previous applications of RPA-LF technology in malaria diagnostics, including the development of species-specific assays detecting four human-infecting species [22][23][24] .While P. falciparum bears the highest disease burden, monitoring all human-infecting species is critical for effective disease surveillance and tailored interventions.In silico analysis confirmed the assay's inclusivity across human-infecting Plasmodium species, essential for optimal performance as primer-template mismatches can otherwise lead to false negatives 25 .Subsequent screening of clinical isolates for P. falciparum, P. vivax, P. malariae, and P. ovale species demonstrated a detection rate of 97.8% (45/46), surpassing the WHO's 97% minimum sensitivity threshold for point-of-need malaria diagnostics 26 .However, further validation is needed through expanded screening of clinical isolates, including P. knowlesi and negative infections, and standardisation of DNA extraction methodologies across all samples.www.nature.com/scientificreports/Additionally, assessing the assay's field applicability and the feasibility of using crude-extraction methods for processing human blood samples directly are essential.

An. stephensi
Our study successfully detected low-density P. falciparum infections below the WHO's 200 parasites per µl 'low parasite density' threshold, as reported in their latest Malaria RDT Performance report 19,27 .The established limit of detection (LoD) for P. falciparum of 10-100 fg is comparable to other RPA-based pathogen assays [28][29][30] , aligning with the sensitivity of other Plasmodium-detecting NAATs such as mini-dbPCR-NALFIA and P. knowlesi RPA-LF, both with a sensitivity of 10 parasites/µl.Early detection of malaria is crucial for improving clinical outcomes 31,32 , highlighting the importance of achieving high sensitivity in our assay.For broader implementation by control programs, reducing the cost of the RPA-LF assay-currently £6.80 per reaction (TwistAmp® Basic 1 × Reaction: £3.87 and Abingdon PCRD LF Cassette £2.93 as of June 2024)-to align with other solutions is imperative.Furthermore, similar to existing Pan Malaria RDTs, positive results would require further speciesspecific screening.
The presence of Plasmodium spp.parasites was also evaluated across three Anopheles spp.Vectors-An.stephensi, An. funestus s.l., and An.gambiae s.l.-affirming the assay's effectiveness as a tool for molecular xenomonitoring surveillance of malaria.Testing reared Anopheles mosquitoes that had recently fed on infected blood demonstrated the capability to detect Plasmodium spp.before the establishment of infection in the mosquito.To our knowledge, this is the first study to demonstrate the detection of Plasmodium spp.using RPA-LF technology in both host and vector isolates.However, further enhancements are needed to align assay sensitivity with current vector screening practices, particularly evident in field samples collected from the DRC.This approach will allow for the detection of Plasmodium infections in vectors after blood meal digestion, regardless of the stage in the sporogonic cycle.Improvements may involve optimising the ratio of reaction mixture loaded onto the lateral flow cassette and increasing the concentration of recombinase proteins that aid in template priming and amplification.Such enhancements would eliminate the need to differentiate between blood-fed and unfed vectors during collection, prior to pooling and screening.This molecular approach is less labour-intensive than  traditional mosquito dissections aimed at detecting sporozoites.Additionally, screening prior to blood meal digestion creates the opportunity to detect a wide range of parasites in hematophagous insects, regardless of their status as vectors.This approach could provide insights into the prevalence of Plasmodium infection within a given collection area.
The ability to operate RPA-based assays using human body heat and incorporate a lateral flow cassette for result detection reduces reliance on specialised equipment compared to other NAATs like LAMP and qPCR.Further validation work is needed to demonstrate the feasibility of using the RPA-LF assay in equipment-free settings with human body heat for incubation.Our presented Pan Plasmodium RPA-LF assay opens pathways for non-specialist use, akin to existing malaria RDTs or lateral flow-based diagnostics for SARS-CoV-2.When combined with crude nucleic acid extraction, this assay offers a point-of-need alternative for disease surveillance programs in resource-limited settings where qPCR facilities are unavailable.Consequently, it eliminates the need to transport and process samples at centralised laboratories, which is standard practice in most surveillance efforts.For instance, the assay could be integrated into ongoing field trapping and morphological speciation efforts to monitor the spread of the invasive An. stephensi species across Africa-a major concern highlighted by the WHO for malaria control.This approach can assess how the distribution of infected vectors influences malaria transmission and guide appropriate intervention strategies 33 .However, similar to other NAAT-based methods for vector incrimination, the methodology's utility should be considered alongside its potential to overestimate infection rates, as it detects any Plasmodium DNA present, not exclusively from infected mosquitoes harbouring sporozoites 11 .
In summary, we have demonstrated the versatile surveillance capabilities of the Pan Plasmodium RPA-LF assay, which, when paired with crude DNA extraction, has the potential to become a pivotal tool in malaria surveillance programs in field settings with further refinement.

Cultured and neglected plasmodium spp.
Plasmodium falciparum (Dd2) and P. knowlesi (A1-H.1)DNA was extracted from parasite cultures, kindly provided by Robert Moon (LSHTM), utilising the DNeasy Blood & Tissue Kit (Qiagen).P. malariae, P. ovale curtisi and P. ovale wallikeri DNA samples were extracted from blood samples from infected travellers returning to the UK, who were diagnosed with malaria between 2019 and 2020, as confirmed by the UK Health Security Agency-Malaria Reference Laboratory at the LSHTM.Speciation of infections was performed by nested PCR and qPCR as outlined previously 34

Plasmodium falciparum
A total of 24 whole blood samples were selected from P. falciparum gametocyte carriers, aged between 5-50 years, recruited into one of the previously published clinical trials in Ouélessébougou, Mali 18,35 .Permission to conduct this study was obtained from the LSHTM Research Ethics Committee (Ref: 17,507) and the University of Sciences Techniques and Technologies of Bamako Ethical Committee (Ref: 2019/67/CE/FMPOS and 2020/96/CE/ FMPOS/FAPH) and performed in accordance with relevant guidelines and regulations.The trial was registered on ClinicalTrials.gov(NCT04049916).Written informed consent was obtained from all subjects and/or their legal guardians prior to sample collection.For minor participants, informed consent for study participation was obtained from their parent and/or legal guardian.Species identification was carried out by microscopy by trained microscopists at the Malaria Research and Training Centre of the University of Bamako (Bamako, Mali).DNA was extracted from 83.3 μl whole blood using a MagNAPure LC automated extractor (Total Nucleic Acid Isolation Kit High Performance; Roche Applied Science, Indianapolis, IN, USA) 18 .

Plasmodium vivax
A total of 12 whole blood samples were selected from P. vivax carriers, aged between 13-30 years, recruited as part of a cohort study of pregnant women, performed in the State of Acre, Brazil, between 2013-2015 36 .Permission to conduct this study was obtained from the Ethics Committee from the University of São Paulo-Brazil (CEP/USP) and the National Commission for Research Ethics (CONEP) (Plataforma Brasil, CAAE nº 32,707,720.0.0000.5467),and performed in accordance with relevant guidelines and regulations.Written informed consent was obtained from all subjects and/or their legal guardians prior to sample collection.Species identification was first performed by microscopy by the endemic surveillance team in the State of Acre and then confirmed by molecular diagnosis using the real-time PCR technique 37 .DNA was extracted from 200 μl whole blood using QIAamp DNA blood mini kit (Qiagen).

Anopheles stephensi-human malaria transmission facility
A cohort of An. stephensi (SD500 strain) were fed with a NF54 P. falciparum asexual laboratory strain culture at approximately 1% parasitaemia, by the LSHTM Human Malaria Transmission facility team.A total of 24 blood fed mosquitoes were randomly selected 4 h post feed, in addition to 6 unfed mosquitos for further pooling and screening.

Field Anopheles spp
A total of 29 Anopheles spp, field isolates were collected in 2018-2019, using indoor Centers for Disease Control and Prevention light traps in two sites in Sud-Kivu province (Tchonka and Tushunguti) and one site in Haut-Uélé province (Kibali), DRC.Following collection, vectors were speciated (An.funestus s.l. or An.gambiae s.l.) by morphology and classified as either blood-fed or unfed, by a trained entomologist.Genomic DNA was

Fig. 2 .
Fig. 2. A subset of the P. falciparum and P. vivax clinical isolates screened using the Pan Plasmodium RPA-LF assay, including the negative P. vivax isolates (V122).Lateral flow cassettes have been cropped to remove the loading port.Lane C (C) represents the flow-check line and Lane 2 (2) detects the FAM/Biotin labelled amplicons generated via the Pan Plasmodium RPA-LF assay.

Fig. 3 .
Fig. 3. Screening of eight infected-blood-fed mosquitoes (AS1-8) and two uninfected An. stephensi mosquitoes (ASN1, ANS2), provided by the Human Malaria Transmission facility.The positive control (PC) and negative control (NC) consisted of P. falciparum and water, respectively.Lateral flow cassettes have been cropped to remove the loading port.Lane C (C) represents the flow-check line and Lane 2 (2) detects the FAM/Biotin labelled amplicons generated via the Pan Plasmodium RPA-LF assay.

Fig. 4 .
Fig. 4. A subset of single (AC) and pools (AP) of infected-blood-fed Anopheles spp mosquitoes screening using the Pan Plasmodium RPA-LF assay following crude extraction.Lateral flow cassettes have been cropped to remove the loading port.Lane C (C) represents the flow-check line and Lane 2 (2) detects the FAM/Biotin labelled amplicons generated via the Pan Plasmodium RPA-LF assay.

Table 5 )
. Across all Pan Plasmodium A B . The UK National Research Ethics Service (Ref: 18/LO/0738) and LSHTM Research Ethics Committee (Ref: 14,710) provided approval for the project.